Determination of the lactose and galactose content of common foods: Relevance to galactosemia

Abstract Classical galactosemia (CG) is a disorder of galactose metabolism which results from deficiency of the enzyme galactose‐1‐phosphate uridylyl transferase (GALT). Treatment consists of immediately eliminating galactose from the diet in the new‐born and lifelong restriction of dietary galactose. The inclusion of a wider variety of foods for people with CG may provide many benefits, including improved nutritional adequacy and quality of life. Galactose plays an important role in glycosylation of glycoproteins and glycolipids. Moderate liberalization of galactose restriction has been shown to improve immunoglobulin G (IgG) glycosylation for some individuals with CG. Moreover, recent outcome research suggests that strict restriction of nondairy galactose may have more unfavorable outcomes than moderate liberalization in CG patients. In the current work, based on patient feedback, we have analyzed the lactose and galactose content of different foods available in Ireland. These include a range of cheeses, yogurts, pizzas, soups, biscuits, cakes, pastries, crackers, mayonnaises, salad creams, fat spreads, crisps, corn chips, salamis, and gravies. This work provides information to support the development of a practical food‐based approach to facilitate analysis of dietary galactose intake and to possibly increase overall variety of food choices for people with CG.

in the Irish Traveler population, with an estimated birth incidence of 1:33,917 in the non-Traveler Irish population (Coss et al., 2013).
Diagnosis is established by measuring galactose or its metabolites such as galactose-1-phosphate (Gal-1-P) and galactitol, in blood or urine. The gold standard for the diagnosis of CG is the measurement of GALT activity in red blood cells (RBCs) (Coelho et al., 2017).
Treatment consists of immediately eliminating galactose from the diet in the new-born and for lifelong strict restriction of dietary galactose. Although this intervention is life-saving in the new-born, there is abundant evidence internationally of long-term complications in treated patients that include cognitive impairment, speech abnormalities, neurological abnormalities, behavior abnormalities, and a very high prevalence of primary ovarian insufficiency in affected females (Colhoun et al., 2018;Fridovich-Keil et al., 2011;Rubio-Gozalbo et al., 2010). The UK Steering Group on Galactosaemia in 1999 proposed that a strictly restricted galactose diet should be followed for individuals affected with CG (Walter et al., 1999). Historically, the diet for children with CG was not as strict at the age they entered the junior school system at approximately 5-6 years old (Francis, 1974). The diet remained free from dairy products, although food products containing traces of lactose/ galactose were consumed in order to manage this diet within a realworld environment.
International clinical guidelines for the management of CG were published in 2017 . The Guideline Taskforce recommended treating CG patients with a lifelong galactose-restricted diet that only eliminates sources of lactose and galactose from dairy products. There was insufficient evidence to support a specific agerelated recommendation for the quantity of galactose allowed in the diet.
This guideline permits galactose from non-milk sources that contribute minimal dietary galactose with the inclusion of small amounts of galactose present in specific mature cheeses and caseinates. It also recommended that any amount and type of fruits, vegetables, legumes, unfermented soy-based products, and the food additives sodium or calcium caseinate could be included in the diet of CG patients .
Galactose plays an important role in glycosylation of glycoproteins and glycolipids (Pucic et al., 2011). Moderate liberalization of galactose diet has been shown to improve immunoglobulin (IgG) glycosylation and to be associated with improved glycosylation in a small group of children and in adults (Coss et al., 2012;Treacy et al., 2021).
Recently, a retrospective dietary analysis of 231 CG patients showed no significant association between non-dairy galactose restriction in early childhood and the outcomes studied (including growth, adaptive behaviors, receipt of speech therapy, or educational assistance) (Frederick et al., 2017). Subjects with a higher galactose intake did not exhibit an increased incidence of complications and those who were very compliant with dietary restrictions did not have more favorable outcomes (Hughes et al., 2009). Outcome studies performed by our own group, and others, have indicated that the severity of dietary galactose restriction is not associated with better cognitive and neurological outcomes. The studies indicated the converse, i.e., that over-restriction of dietary galactose might be harmful (Hughes et al., 2009;Jumbo-Lucioni et al., 2012), with further evidence suggesting that strict restriction of non-dairy galactose may possibly have sub-optimal rather than beneficial outcomes (Rubio-Gozalbo et al., 2019;Schweitzer et al., 1993;Waisbren et al., 2012). In the recently reported GalNet (The Galactosemia Network) registry outcome study of over 500 CG patients, it was noted that patients following a strict galactose-restricted diet (lactose, fruit, and vegetables restricted) developed neurological complications more frequently than patients with a less strict diet (Rubio-Gozalbo et al., 2019).
Galactose is derived from the breakdown of lactose. Lactose is the disaccharide of milk and milk-containing products. Lactose is hydrolyzed to glucose and galactose by lactase in humans (Reichardt & Berg, 1988;Valle et al., 2019). During the process of digestion, lactose is broken down in the ratio of 47.37% glucose:52.63% galactose. Therefore, 1 g or 1000 mg lactose = 526 mg ~ 500 mg galactose by extrapolation (Ohlsson et al., 2017). On a galactose-restricted diet, endogenous galactose production ranges from 1.1 to 1.3 g/ day (Liu et al., 2000). It has been estimated that the rate of de novo synthesis of galactose, in healthy and in subjects with galactosemia, ranges from 0.48 to 1.71 mg/kg/h (Berry et al., 2004). For an average adult, weighing 70 kg, this is equivalent to 800-2873 mg galactose per day. Endogenous production of galactose was not found to be affected by the exogenous intake of lactose and galactose from the diet (Huidekoper et al., 2005).
It has also been shown that the daily ingestion of 200 mg of fruit-derived galactose had no impact on RBC Gal-1-P values and relatively little effect on urinary galactitol levels in patients with null RBC GALT activity (Coelho et al., 2017;Berry et al., 1993). This may suggest that endogenous galactose production is the main source of galactose metabolites routinely detected in patients on lactoserestricted diets (Berry et al., 1993). Subsequently, it has been noted that there is significant endogenous galactose production in children and adults (Berry et al., 2004;Schadewaldt et al., 2004). Thus, foods such as offal, fruits, legumes, and pulses are insignificant sources of galactose compared with endogenous production and there is no evidence to support their restriction (Berry et al., 1995). Furthermore, a number of commonly consumed foods have been found to have reduced galactose content following analysis and have therefore been suggested for inclusion in a galactose-restricted diet. These include lactose-hydrolyzed milk fermented using a traditional kefir culture (Varga et al., 2006), Pecorino Romano sheep cheese (Idda et al., 2018), Gruyere, Emmental, Jarlsberg, Italian Parmesan (Parmigiano Reggiano and Grana Padano), Comte, Emmi Swiss Fondue, and specific British brands of mature cheddar cheese. However, the galactose content of processed foods can vary significantly as thermal changes, clarification aid, and enzymatic liquefaction aid may contribute to altered galactose content during food processing (Scaman et al., 2004).

Significant improvement in proteins and lipids glycosylation
was demonstrated with moderate galactose diet liberalization in a subset of adults and children, specifically allowing up to 500 mg of galactose in the pediatric study and with beneficial glycosylation effects up to 1000 to 2000 mg of galactose in adults (Lee et al., 2003;Maratha et al., 2017;Panis et al., 2006). Bosch et al., in a study of adolescents with galactosemia, noted that a daily dietary intake of 500 mg of galactose was well tolerated (Bosch, 2011).
There are also reports on individual adult CG patients who selfliberalized their diets without adverse consequences. No physical, ophthalmological, or biochemical abnormalities were detected in three adolescents who ingested up to 600 mg of galactose per day for 6 weeks (Bosch et al., 2004). When these cases were compared to adherent patients, they reported a better quality of life (Lee et al., 2003;Panis et al., 2006).

| Patient surveys
An anonymous patient questionnaire was sent to all CG patients or their carers attending the National Centre for Inherited Metabolic Disorders, Temple Street Hospital, Dublin in 2012. This included a request to list foods that patients would like analyzed to ascertain if these products contained sufficiently minimal amounts of galactose to be allowed in the diet. Information from this questionnaire was used to perform a phone survey involving 13 adults and 8 children with CG in 2015 (Appendix S1). Informed verbal consent was obtained from all patients prior to administering the questionnaire.

| Chemical analysis of lactose and galactose in the food samples using high-performance anion-exchange chromatography/pulsed amperometric detection
Food selected for analysis from the phone survey were all analyzed between 2016 and 2021, at the Public Analyst's Laboratory, Galway, using high-performance anion-exchange chromatography/pulsed amperometric detection (HPAEC/PAD) to accurately determine galactose and lactose concentrations in food products. This analytical method is an in-house developed method and is accredited to ISO 17025 since November 2019. The analysis accurately determines sucrose, galactose, glucose, fructose, lactose, and maltose concentrations in beverages and foods. Food brand names were anonymized and individual samples were labeled by a laboratory reference number in all results tables (see Appendix S1). Food samples were mixed, homogenized, or grated (in the case of hard cheese finely grated) before analysis. A test portion was extracted with carrez solution. Some food matrices, i.e., pizza and cheese etc. required an Ultra-Turrax step with water prior to the carrez extraction step and emulsified samples (i.e., butters, spreads, mayonnaises, etc.) were de-emulsified prior to the carrez extraction step, by placing a test portion with water in a water bath set at 45°C for 5-30 min. The sample extract was vortexed for ~15 s to thoroughly mix the contents and centrifuged at 3000 rpm for 20 min at ambient temperature and filtered.

| Patient survey
The results of the phone survey were reviewed by two clinical dietitians and the following food groups were chosen for analysis;

| HPAEC/PAD method analytics
Spiking recovery studies were carried out for all food types.

| Food analysis
Results are presented in ascending total galactose content (mg/100 g) for all food groups. Minimum and maximum values for galactose, lactose, "released" galactose, total galactose, portion size, and total galactose per portion are included for each food type. Table 1 provides an overview of all foods analyzed. The analysis of dairy and dairy alternatives included 35 cheeses (Table S4a) two Dutch semi-hard cheeses including a popular miniature snack cheese, and four non-cheddar white cheeses including two reduced fat white cheeses and one vintage. Of the six mature cheddars with galactose <25 mg/100 g, two were of the same type and brand but from different batches. The remaining six samples from this cheese had galactose contents ranging from 42 to 255 mg galactose per 100 g. The remaining seven mature cheddar cheeses had galactose contents ranging from 32 to 237 mg/100 g. Two non-mature cheddar cheeses from one brand were analyzed and were shown to have galactose contents of 193-283 mg/100 g. The cheeses with the highest galactose content were cheeses labeled "lactose-free" (824-908 mg/100 g) and processed cheeses including a soft cheese block and cheese slices (721--2656 mg/100 g). This analysis confirms the suitability of dairy-free cheeses and the unsuitability of both lactose-free and highly processed cheeses/cheese spreads in a galactose-restricted diet. Some mature cheddar cheeses were found to have minimal galactose content (<25 mg/100 g). However, the majority had a higher galactose content. A number of cheeses for which no previous analyses exist were included in this study, including two types of Dutch semi-hard cow's milk cheese, reduced fat white cheeses, and one vintage cheese from a popular Irish brand. All of these cheeses had low galactose contents (<25 mg/100 g).
Twenty-three yogurt products were analyzed (Table S4b). All non-dairy yogurts labeled "dairy-free" (coconut and soya-based yogurts) contained <50 mg galactose per 100 g and all dairy-containing yogurts contained >1600 mg galactose/100 g. This analysis confirms the suitability of dairy-free yogurts for patients avoiding galactose and provides more insight into the galactose contents of a wide range of dairy-containing yogurts. As all dairy-containing yogurts contained a significant amount of galactose, they remain unsuitable for most patients on a galactose-restricted diet, unless included in very small, measured quantities as part of a galactose liberalized diet. Table S4c,d outlines the analysis of 16 supermarket-bought pizzas which were cooked prior to analysis. All pizzas contained dairy cheese and ranged in galactose content from 51 to 216 mg per 100 g.  No milk declared/may contain (traces of) milk/produced in a factory handling milk but on a different line/manufactured on equipment that handles milk).

TA B L E 1 (Continued)
Pepperoni and ham & pineapple topped pizzas had a slightly lower galactose content than cheese-only pizzas (51-177 mg compared to 71-216 mg per 100 g). The regular cheese pizza, pepperoni pizza, and ham & pineapple pizza from four popular franchise restaurants were also analyzed.
All franchise pizzas contained dairy cheese and ranged in galactose content from 132 to 350 mg per 100 g.
Pepperoni and ham & pineapple topped takeaway pizzas also had a slightly lower galactose content than cheese-only takeaway pizzas (132-308 mg compared to 168-350 mg per 100 g) ( Table S4d).
Thus, all supermarket-bought pizzas analyzed contained <400 mg galactose per portion (half a pizza). Takeaway pizzas tended to have higher galactose contents and are likely to be less standardized than supermarket-bought pizzas.
Our results also indicate that cheese pizzas are higher in galactose compared to pizzas with varied toppings (pepperoni, ham & pineapple). Table S4e outlines the lactose and galactose contents of a range of soups (fresh and canned). All dairy-free soups contained minimal galactose (3 mg galactose per 100 g) and soups containing butter and cream as the only milk ingredients had a galactose content of 37-121 mg per 100 g. Soups also containing other milk ingredients contained 84-855 mg galactose per 100 g, including "cream of" soups which ranged from 237 to 282 mg galactose per 100 g for cream of tomato (n = 3), 409-569 mg galactose per 100 g for cream of chicken (n = 2), and 855 mg galactose per 100 g for cream of mushroom (n = 1). Therefore, for patients advised to avoid galactose, dairyfree soups are suitable, as all contained trace amounts of galactose.
Soups containing butter and cream may be suitable for patients on a liberalized diet, as a serving size (400 g) would typically provide 148-484 mg of galactose. However, soups containing other milk ingredients and any soups labeled "cream of" contained higher amounts of galactose and would be unsuitable for patients with CG.
Fourteen types of biscuits were analyzed ( Chocolate-containing biscuits had substantially higher galactose contents compared to plain versions and would therefore be unsuitable for inclusion in a galactose-restricted diet. Six types of crackers from five popular brands were analyzed (Table S4g). Five contained no dairy products and had low galactose contents (≤34 mg per 100 g). Per 100 g, oatcakes had the lowest galactose content (<3 mg), followed by salted savory snack biscuits (5 mg), cream crackers (10 mg), crackerbread (12 mg), and Three popular brands of light and "real" mayonnaise were analyzed (Table S4i). Two light mayonnaises contained milk cream powder and had a higher galactose content of 82-84 mg/100 g, compared to <38 mg galactose per 100 g for all non-dairy containing mayonnaises. Two brands of salad cream were also analyzed (Table S4i). Neither brand contained any milk ingredients but both contained <38 mg galactose/100 g. Nine fat spreads including dairyfree spreads, buttermilk-containing spreads, and pure butter were analyzed (Table S4j). A dairy-free baking block for cakes and pastry contained <15 mg galactose per 100 g and the galactose content of dairy-containing spreads ranged from 12 to 333 mg per 100 g, with pure butter containing the most galactose (333 mg per 100 g or 33 mg per 10 g portion).
Twenty-one types of potato crisps and tortilla/chips were analyzed (Table S4k) Four types of salami, one each from four major supermarket brands, were analyzed ( Table S4l). None of the salami products contained any milk products and all had minimal galactose contents (<8 mg per 100 g). Nineteen types of gravies from six brands were analyzed (Table S4m). Fourteen gravies had no milk ingredients and five contained milk, milk ingredients, lactose, and/or milk proteins.
Eighteen gravies had minimal galactose content (<8 mg per 100 g) and one gravy had 288 mg galactose per 100 g. However, per 100 ml portion, there was only 22.2 mg galactose.

| DISCUSS ION
A galactose-restricted diet has been the mainstay of therapy for management of galactosemia since 1935. The principle of dietary management of CG should be not only to restrict intoxication of galactose and its by-products but also to allow enough substrate for normal growth and development while optimizing nutritional status.
There is still an ongoing debate about the degree of restriction required in childhood and adulthood. Many patients with galactosemia, regardless of the degree of galactose restriction, are still at risk of developing long-term complications, including cognitive delay, language impairment, reduced bone mass, and female infertility. Overrestriction of galactose may contribute to the disease phenotype in susceptible individuals by further depleting uridine diphosphate (UDP)-galactose, and disrupting glycosylation-dependent pathways.
Build-up of galactose-1-phosphate (Gal-1-P) and its metabolites is proposed to contribute to the development of CG complications (Colhoun et al., 2018).
Numerous methods are routinely applied for the detection of mono-and disaccharides in human food including spectroscopy, polarimetry, gravimetry, chromatographic, and enzymatic techniques.
The performance of the HPAEC/PAD system used in the current study showed acceptable spiking recoveries for galactose and lactose for all matrices in the range 85-112% (Appendix S1 - Table   S2). These figures are comparable to recently published data using HPAEC/PAD methods for quantification of fermentable oligo-, di-, and monosaccharides in cereals and cereal-based products (Ispiryan et al., 2019).
When comparing our analysis results with lactose and galactose values in different foods, as cited in McCance and Widdowson (6th Edition) (Paul et al., 1986;Paul & Southgate, 1979) and other analyses (Portnoi & MacDonald, 2015), our results were comparable, particularly in terms of food products such as butter.
Cheese was prioritized for testing, as this patient group is at risk of low bone mineral density and may have suboptimal intake of calcium on a galactose-restricted diet (van Erven et al., 2017).
Furthermore, this analysis aims at validating and consolidating previous analyses of the galactose content of cheeses (Portnoi & MacDonald, 2009;Portnoi & MacDonald, 2016). Our analyses showed that all six dairy-containing noncheddar hard cheeses that were not labeled "lactose-free" had a low galactose content (total galactose <25 mg/100 g), while 35% (6 of 17) of mature cheddar cheeses were low in galactose.
The lactose and galactose content of cheese is variable due to differing manufacturing processes, the specific starter bacterial cultures used, and the extent of cheese maturation.
Lactose and galactose content in cheese is reduced through two processes: the separation and removal of whey and the fermentation of lactose by bacteria (Portnoi & MacDonald, 2009;Portnoi & MacDonald, 2016). The latter process, often accelerated through the addition of a starter bacteria culture, occurs via the lactase in lactic acid bacteria metabolizing lactose and, in some cases, converting galactose to glucose (Portnoi & MacDonald, 2009;Portnoi & MacDonald, 2016). Fermentation periods and the specific bacterial cultures used affect the lactose content and subsequent galactose content postbacterial metabolism the longer the cheese has matured or ripened. In general, this results in a reduced lactose and galactose content following established methods of hard cheese production (Cogan et al., 1998). Different cheese manufacturers will use different methods, bacterial cultures, and maturation times which all contribute to this variability and which can also change over time if new processing methods are introduced.
For soft and processed cheeses, the curd is separated from the whey and then used immediately (Portnoi & MacDonald, 2009;Portnoi & MacDonald, 2016). Therefore, these cheeses contain a relatively high level of galactose. Milk products (including cheese) labeled "lactose-free" are in fact lactose-hydrolyzed milk whereby the lactose is enzymatically converted to glucose and galactose. These cheeses had low lactose content but a much higher galactose content compared to nonhydrolyzed products, as expected. While the processing methods of the cheddar cheeses labeled "lactose-free" were not available, neither cheese was labeled mature.
The variability in the galactose content of the mature cheddar cheeses analyzed may also reflect differences in processing and packing techniques. More traditional manufacturing processes provide the lowest levels of lactose and galactose, while large-scale manufactured cheeses are often packed soon after production, with maturation occurring within the package. With the latter method, lactose is not lost within the package and therefore the galactose content of these cheeses tends to be higher (Portnoi & MacDonald, 2016).
Similar analyses in the United Kingdom have expanded the range of cheeses allowed on a low galactose diet (Portnoi & MacDonald, 2009;Portnoi & MacDonald, 2016). These studies also highlight the importance of the manufacturing process to the lactose and galactose content of cheeses, with certain brands of mature cheddar cheeses excluded due a lactose/galactose content >10 mg/100 g. This analysis may further expand the number of suitable cheeses available to people with CG, to include specific brands of mature cheddar cheese, two types of Dutch semihard cow's milk cheese, reduced fat white cheeses, and one vintage cheese from a popular Irish brand.
Similar to cheeses, milk and yogurt products that have been fermented have a lower galactose content (Ohlsson et al., 2017), with longer fermentation times resulting in lower galactose contents (Varga et al., 2006). As manufacturing methods and fermentation periods can vary widely, further analyses are required to ascertain the galactose content of fermented milk products available on the Irish market and their suitability for inclusion in a galactoserestricted diet.
Whole foods, defined as natural products such as butter and milk, are less likely to experience changes in lactose and extrapolated galactose content over time. In comparison, a more complex food item such as a biscuit or cake containing multiple ingredients and where individual recipes can change without notification to the consumer will be difficult to monitor in terms of estimated lactose and extrapolated galactose content. Furthermore, the higher than expected galactose content of certain products labeled "dairy-free" indicates the potential impact of cross contamination.
Therefore, it is important to note that it is difficult to know the exact and consistent galactose content of individual processed foods due to the variation in ingredients over time and indeed even within the same batch. However, by reviewing the data available, certain foods could possibly be considered for inclusion in a galactose-restricted diet in measurable quantities under metabolic clinic supervision with adequate education. These include specific cheeses, packaged pizzas, soups, biscuits, crackers, cakes, pastries, fat spreads, and crisps.
The inclusion of a wider variety of foods for patients with CG could have many benefits, including improved quality of life and nutritional benefits, e.g., inclusion of foods high in calcium and vitamin D. In particular, the consumption of dairy products is recommended to optimize bone health and reduce the risk of osteoporosis (Wallace et al., 2020). Furthermore, this analysis indicates that butter and dairy-containing spreads, using portion sizes in line with healthy eating guidelines (Flynn et al., 2012;HSE, 2016), may be suitable for CG patients who consume a more liberalized diet. The inclusion of a wider variety of fat spreads in a galactoserestricted diet would greatly increase food choices, particularly when eating out, and may contribute to patients' dietary variety and quality of life.
The results of our analyses describe the galactose contents of a wide range of foods, thereby contributing to our understanding of the nutritional content of processed foods and providing clinicians with further guidance on the suitability of certain foods for inclusion in a galactose-restricted diet. Of note, foods chosen for analysis were based on patient preferences and many of these foods were convenience foods high in fat, sugar, and/or salt while some did not contain any lactose-containing ingredients.
As such, this analysis does not reflect a healthy, balanced diet, nor is it a comprehensive investigation of foods potentially high in galactose. Therefore, patient education and advice regarding diet liberalization should aim to ensure a healthy, balanced diet is consumed.
The results of the present study may provide a useful tool to dietitians to aid patients to make more informed choices on food consumption (particularly processed food intake) and dietary pattern. These results could be used to implement advised increases to dietary galactose intake for suitable patients, with close clinical and dietary monitoring.
To allow the safe introduction of slight increases of galactose in the diet, sensitive biomarkers are required Treacy et al., 2021), with improved analysis of galactose content in common foods.

| CON CLUS ION
This study has enabled the further analysis of food products which could expand the food choice available to patients with CG. This food analysis will be used to develop patient resources which can be individualized to the specific dietary needs of each CG patient.
Improved knowledge of the composition of foods and accurate estimation of food ingredients could provide a powerful tool to advise patients on more varied and balanced dietary intake. A limitation of the study is that the analysis of food samples was based on patient preference only. Furthermore, there are some difficulties in terms of variability of results over time related to the dynamics of an evolving food market, e.g., introduction of new brands, discontinuation of other brands, and modification of portion size or content.

ACK N OWLED G M ENTS
We are particularly grateful to our patients and their families.

CO N FLI C T O F I NTE R E S T
All authors declare that they have no conflict of interest.